Morpholino artifacts provide pitfalls and reveal a novel role for pro-apoptotic genes in hindbrain boundary development
Morpholino antisense oligonucleotides (MOs) are widely used as a tool to achieve loss of gene function, but many have off-target effects mediated by activation of Tp53 and associated apoptosis. Here, we re-examine our previous MO-based loss-of-function studies that had suggested that Wnt1 expressed...
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Veröffentlicht in: | Developmental biology 2011-02, Vol.350 (2), p.279-289 |
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Zusammenfassung: | Morpholino antisense oligonucleotides (MOs) are widely used as a tool to achieve loss of gene function, but many have off-target effects mediated by activation of Tp53 and associated apoptosis. Here, we re-examine our previous MO-based loss-of-function studies that had suggested that Wnt1 expressed at hindbrain boundaries in zebrafish promotes neurogenesis and inhibits boundary marker gene expression in the adjacent para-boundary regions. We find that Tp53 is highly activated and apoptosis is frequently induced by the MOs used in these studies. Co-knockdown of Tp53 rescues the decrease in proneural and neuronal marker expression, which is thus an off-target effect of MOs. While loss of gene expression can be attributed to cell loss through apoptotic cell death, surprisingly we find that the ectopic expression of hindbrain boundary markers is also dependent on Tp53 activity and its downstream apoptotic effectors. We examine whether this non-specific activation of hindbrain boundary gene expression provides insight into the endogenous mechanisms underlying boundary cell specification. We find that the pro-apoptotic Bcl genes puma and bax-a are required for hindbrain boundary marker expression, and that gain of function of the Bcl-caspase pathway leads to ectopic boundary marker expression. These data reveal a non-apoptotic role for pro-apoptotic genes in the regulation of gene expression at hindbrain boundaries. In light of these findings, we discuss the precautions needed in performing morpholino knockdowns and in interpreting the data derived from their use.
► Previous studies have shown that antisense morpholino oligonucleotides (MOs) can have off-target effects mediated by activation of Tp53 and associated apoptosis. ► We show that in the zebrafish hindbrain, MOs can also non-specifically induce ectopic boundary marker gene expression. ► The induction of ectopic gene expression by MOs is due to a normal non-apoptotic role of the Bcl–caspase pathway that is required for hindbrain boundary gene expression. ► These findings reveal that a previous study suggesting that Wnt1 promotes neurogenesis and restricts hindbrain boundary marker expression is incorrect. ► The finding that MOs can induce non-apoptotic roles of the pro-apoptotic pathway further emphasizes the need for precautions in their use and interpretation of results. |
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ISSN: | 0012-1606 1095-564X |
DOI: | 10.1016/j.ydbio.2010.11.030 |